Pharmacologic method of lowering cholesterol production
View Patent ↗The present invention provides a screening method for identifying test agents that modulate cell membrane cholesterol activity. The modulating activity of the test agents may be measured using lytic compounds, which cause cell lysis or increases in cell permeability in response to cell membrane cholesterol levels. The invention further provides therapeutic agents that are identified using the screening method. The therapeutic agents either increase or decrease the cell membrane cholesterol activity, cholesterol concentration and/or both in vivo and/or in vitro.
1. A method of screening compounds for cholesterol modulating activity, comprising:
(a) providing an array that comprises two or more samples of the same type of cell in vitro under physiological conditions;
(b) contacting one or more test agents with the cells;
(c) determining whether the one or more test agents increase cholesterol activity in the plasma membrane of the cells; and
(d) selecting those test agents that increase cholesterol activity, and determining if the selected test agents reduce HMG-CoA reductase and LDL receptor levels in cultured cells of the same cell type as the cells of steps (a)-(c).
2. The method of claim 1 , wherein (c) comprises contacting the cells with lytic compound, wherein the lytic compound causes perforation or lyses of the membrane of the cells when the cholesterol activity of the membrane of the one or more cells reaches a level at or above a threshold cholesterol level.
3. The method of claim 2 , wherein the lytic compound comprises a polyene antibiotic.
4. The method of claim 2 , wherein the lytic compound comprises a lysophosphatide or cholesterol oxidase.
5. The method of claim 2 , wherein the lytic compound comprises a bacterial toxin.
6. The method of claim 1 , further comprising performing (b) and (c) one or more times with different test agents.
7. The method of claim 6 , wherein the different test agents are screened simultaneously.
8. The method of claim 1 , wherein the cells comprise one or more eukaryotic cells.
9. The method of claim 8 , wherein the one or more eukaryotic cells comprise one or more mammalian cells.
10. The method of claim 8 , wherein the one or more cells comprise one or more red blood cells.
11. The method of claim 8 , wherein the one or more cells comprise one or more fibroblasts.
12. The method of claim 8 , wherein the one or more cells comprise one or more human cells.
13. The method of claim 1 , wherein the one or more cells have vigorous cholesterol homeostasis.
14. The method of claim 1 , wherein (c) comprises measuring the permeability of the membrane of the one or more cells or the turbidity of the one or more cells.